Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F21%3A00548916" target="_blank" >RIV/86652036:_____/21:00548916 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0045206821008026?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0045206821008026?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.bioorg.2021.105425" target="_blank" >10.1016/j.bioorg.2021.105425</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher

Popis výsledku v původním jazyce

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the epsilon-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted super-substrates with catalytic constants of up to 15,000,000 M(-1)s(-1) for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD(+) has IC50 values below 200 pM.

Název v anglickém jazyce

Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)-based fluorescence quencher

Popis výsledku anglicky

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the epsilon-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted super-substrates with catalytic constants of up to 15,000,000 M(-1)s(-1) for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD(+) has IC50 values below 200 pM.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10401 - Organic chemistry

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Bioorganic Chemistry

ISSN

0045-2068

e-ISSN

1090-2120

Svazek periodika

117

Číslo periodika v rámci svazku

DEC 2021

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

14

Strana od-do

—

Kód UT WoS článku

000711454100003

EID výsledku v databázi Scopus

2-s2.0-85117796745